MLN and spleen single-cell suspensions were prepared for flowcytometry analysis as previously described [61 (link)]. The cell suspensions were incubated with anti-mouse CD16/CD32 (Mouse BD Fc Block; BD Biosciences, Franklin Lake, NJ, USA) in PBS + 1% BSA for 15 min on ice to block unspecific binding sites. Afterwards, cells were stained with the following surface markers CD4-PerCp-Cy5.5, CD69-PE-Cy7, CXCR3-PE, CD25-AlexaFluor488, CD25-PE, (all purchased from eBioscience, San Diego, CA, USA) or T1ST2-FITC (MD Bioproducts, St. Paul, MN, USA) for 30 min on ice. Fixable Viability dye eFluor 780 (eBioscience) was used to exclude non-viable cells. Next, the cells were fixed and permeabilised with the Foxp3 Staining Buffer Set (eBioscience) according to the manufacturer’s protocol and then stained with the intracellular markers Foxp3-PE-Cy7, RORɣt-PE, IRF4-FITC, Tbet-eFluor660 or Gata3-eFluor660 (all purchased from eBioscience). Cells were measured on BD FACSCanto II flow cytometer, and results were analysed with FlowLogic software (Inivai Technologies, Mentone, Vic, Australia). The used gating strategy is shown in Figure S1 .
Cd69 pe cy7
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CD69-PE-Cy7 is a fluorescently labeled antibody used in flow cytometry applications to detect and quantify the expression of the CD69 cell surface antigen. CD69 is an early activation marker for various immune cell types. The PE-Cy7 fluorescent dye conjugated to the CD69 antibody allows for the identification and analysis of activated cells expressing this marker.
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Lab products found in correlation
Cd25 pe, by Thermo Fisher Scientific (3 mentions)
Lsrfortessa, by BD (3 mentions)
Lsrii flow cytometer, by BD (3 mentions)
Tbet efluor660, by Thermo Fisher Scientific (2 mentions)
Mouse fc block, by BD (2 mentions)
Cd4 percp cy5, by Thermo Fisher Scientific (2 mentions)
Cd25 alexafluor488, by Thermo Fisher Scientific (2 mentions)
Cxcr3 pe, by Thermo Fisher Scientific (2 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (2 mentions)
Flowlogic software, by Inivai Technologies (2 mentions)
Efluor 780, by Thermo Fisher Scientific (2 mentions)
Cd62l percp cy5.5 mel 14, by Thermo Fisher Scientific (2 mentions)
Lag 3 pe c9b7w, by Thermo Fisher Scientific (2 mentions)
Tim 3 pe 8b 2c12, by Thermo Fisher Scientific (2 mentions)
Pe cy7 pd 1 29f 1a12, by BioLegend (2 mentions)
Tigit apc 1g9, by BioLegend (2 mentions)
C maf efluor660, by Thermo Fisher Scientific (2 mentions)
Ki67 pe, by Thermo Fisher Scientific (2 mentions)
Foxp3 pe cy7, by Thermo Fisher Scientific (2 mentions)
Cd4 apc, by Thermo Fisher Scientific (2 mentions)
20 protocols using cd69 pe cy7
1
Multiparameter Flow Cytometry Analysis
2
Isolation and Characterization of Liver Immune Cells
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The liver nonparenchymal cells were isolated as described [18] (link). The harvested cells were stained with two separate panels of antibodies, including CD3-PE (#553,063, BD Bioscience), CD8-FITC (#553,031, BD Bioscience), CD69-PE-Cy7 (#25-0691-81, eBioscience), CD45-APC—Cy7 (#103,116, Biolegend) and CD11b-PE-Cy7 (#101,215, BioLegend), TNF-α-Pacific Blue (#506,318, Biolegend), Ly6G-Percp-Cy5.5 (#127,616, Biolegend).
3
Multiparametric Immune Phenotyping
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Stimulated cells were incubated with Fcγ receptor blocking antibody for 20 min on ice and then stained for 30 min on ice with fluorophore-conjugated antibodies: CD56-APC, CD69-PEcy7, Icam-1-PE, CD86-Alexa Fluor 488, CD11b-FITC, CD66b-APC and CD16-PE, from eBioscience and LFA1-PECy5, CD14-APC/Cy7 and DNAM-1-FITC from Biolegend. After washing thrice with PBS, the expression of surface molecules was measured by flow cytometry (Beckman CyAn and BD BioScience Fortessa) and data were analyzed with Flow Jo software and Summit software. The mean fluorescence intensity from specific antibodies was obtained by subtracting the values from control isotype antibodies. Lymphocytes, monocytes, and granulocytes, amongst the WBC population, were gated based on side scatter and forward scatter, which were not affected by stimulation (Supplementary Fig. 1 ).
4
Selective Kv1.3 Channel Blocking by ShK-223
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ShK-223, an analog of sea anemone peptide ShK, was used as a highly selective Kv1.3 channel blocker (IC50 for Kv1.3 25 ± 14 pM, >10,000-fold more selective for Kv1.3 than neuronal Kv1.1 and Kv1.2 channels) [12 (link)]. The dose of ShK-223 for intravenous or intraperitoneal injections was 100 μg/kg. Concentration used for in vitro experiments was 100 nM. ShK-F6CA, a fluorescein-conjugated ShK analog, was purchased from Peptides International (Louisville, KY). LPS was obtained from Sigma-Aldrich (Cat #L4391, Escherichia coli 0111:B4). Fluorophore-conjugated monoclonal antibodies for flow cytometry were obtained from BD Biosciences (CD11b-PeCy7 or APC-Cy7, CD45-PerCP or PE-Cy7, ICAM1-APC, CD69-PECy7, pS727-STAT1-Alexa647), eBiosciences (CD3-FITC, CD8-APC, CD95-PE) and Cell Signaling Technologies (pY701-STAT1-Alexa488).
5
PBMC Immunophenotyping by Flow Cytometry
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For the CD4+ cells or PBMCs analysis, harvested cells were resuspended in 50 µl of fluorescent-activated cell sorting buffer and stained for 30 min on ice for respective membrane antigens using fluorochrome conjugated monoclonal antibodies specific for human: CD4–FITC, CD25-PE, human leukocyte antigen (HLA-) DR-PerCP (all from BD Pharmingen, San Diego, CA, USA), CD8-PerCP, and CD69-Pe-Cy7 (both from eBioscience, San Diego, CA, USA). To assess viability, PBMCs were stained with 7-aminoactinomycin D (7AAD). After the washing step, cells were acquired and analyzed using a FACSCanto cell cytometer and Diva software. Appropriate isotype controls were used in all experiments. The gating strategy and applied isotype controls are shown in Supplemental Fig. S1.
6
Phenotyping of Natural Killer Cells
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Antibodies and other reagents used were: Intracellular Fixation and Permeabilization Buffer kit, fixable viability dye e-Fluor 780, CD56 PerCP-eFluor 710, MICA/B Alexa Fluor 488, CD69 PE-Cy7 (eBioscience, Grand Island, NY, USA); CD16 PE, CD56 PE (BD Biosciences, San Jose, CA, USA); CD3 FITC, IFN-γ PE-Vio770, TNF-α PE-Vio770, CD107a FITC (Miltenyi Biotec, Auburn, CA, USA); CD3 PE-Cy7, CD3 FITC, CD56 APC (MY31) (Tonbo Bioscience, San Diego, CA, USA); perforin Pacific Blue, granzyme B Pacific Blue, NKG2D APC (BioLegend, San Diego, CA, USA); and survivin Alexa Fluor 647 (Cell Signaling Technology, Danvers, MA, USA). Data were collected on a Miltenyi MACSQuant Analyzer flow cytometer (Miltenyi Biotec, Auburn, CA, USA) and analyzed using FlowJo v10.0.8p software (Tree Star, Ashland, OR, USA).
7
Evaluating Dendritic Cell Surface Markers
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Expression of DC surface markers was evaluated by LSR-II cytometer (BD Bioscience) using the following antibodies: anti-MHC class II-PE-Cy7 (#107629/BioLegend) or APC-Cy7 (#107628/BioLegend) anti-CD11c-FITC (#117306/BioLegend) or PerCP-Cy5.5 (#560584/BD Pharmingen), anti-CD86-PerCP (#105025/BioLegend), anti-CD40-Pacific Blue (#124626/BioLegend), anti-PD-DL-1-Qdot-605 (#124321/BioLegend), ICAM-1-FITC (#11054182/eBioscience), CD18-PE (#101408/BioLegend), CD69-PE-Cy7 (#25069182/eBioscience), CD11a-APC (#101120/BioLegend), CD44-e450 (#48044182/eBioscience), CD11b BV605 (#83011242/eBioscience; all used in 1:100 dilution), and anti-AXL-APC (#FAB8541A/R&D Systems; dilution 1:20). The dead cells were excluded with Fixable Viability Dye eFluor 780 or e506 (#650866/ThermoFisher Scientfic). There was no difference in viable cell proportion of BM-derived DCs between WT and miR-34a−/− genotypes (mean±s.d: 75% ±12 and 71% ±10, respectively).
8
Isolation of Immune Cells from Murine Tissues
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Cells from the aorta, spleen and paLN were isolated as described before19 (link). Briefly, Apoe−/−Il27ra+/− or Apoe−/−Il27ra−/− mice were euthanized by CO2 inhalation. The aortas were perfused with 30 ml of PBS containing 2% heparin and isolated under the dissection microscope. Collected aortas, spleens or paLNs were cut into small pieces followed by digesting in 2 ml of enzymatic cocktail, containing 450 U/ml collagenase type I, 250 U/ml collagenase type XI, 120 U/ml hyaluronidase type I, 120 U/ml DNAse I (all enzymes from Sigma) in 1x HBSS and incubated in a shaker at 37 °C for 55 min. Obtained cell suspension was stained with following antibodies: CD45-PerCP (30-F11; BioLegend), CD11b-eFluor 450 (M1/70; eBioscience), CD11c-APC (N418; eBioscience), MHCII-Alexa Fluor 700 (M5/114.15.2; eBioscience), TCRβ-eFluor 780 (H57-597; eBioscience), CD69-PE-Cy7 (H1.2F3; eBioscience), B220-FITC (RA3-6B2; eBioscience), CD4-APC (GK1.5; eBioscience), CD8-PE (53-6.7; eBioscience) and LIVE/DEAD Yellow fixable dye (Invitrogen) and analyzed by flow cytometry (LSRII, BD Biosciences). Obtained data were analyzed using FlowJo software.
9
Flow Cytometry Analysis of Immune Cell Subsets
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Cells were analyzed on FACSCantoII (BD) and FlowJo software (Tree Star), and sorted on FACS Aria II (BD). Antibodies used were CD122 PerCP-eFluor 710 (TM-b1), CD19-PerCP-Cy5.5 (eBio1D3), CD24-FITC (M1/69), CD25-PE (PC61.5), CD27-APC (LG.7F9), CD3ε-V500 (500A2), CD4-APC-eFluor 780 (RM4-5), CD44-FITC/APC/APC-eFluor 780 (IM7), CD45.1-PerCP-Cy5.5 (A20), CD45.2-APC-Cy7 (104), NK1.1-PE-Cy7/PerCP-Cy5.5 (PK136), CD62L-FITC/APC/PE-Cy7 (MEL-14), CD69-PE-Cy7 (H1.2F3), CD8α-APC (53–6.7), βTCR-APC-eFluor 780 (H57-597), Qa-2 FITC (69H1-9-9), and γδTCR PerCP-eFluor 710 (eBioGL3; all from eBioscience). iNKT cells were stained at room temperature using α-GalCer–loaded CD1d tetramers. Stainings for intracellular antigens were performed using the FoxP3/Transcription Factor staining buffer set (eBioscience). Active caspase-3+ iNKT cells were stained using PhiPhiLux G1D2 (Oncoimmunin) according to the manufacturer’s recommendations. Subsets are defined as DN1 (CD25−CD44+); DN2 (CD25+CD44+); DN3 (CD25+CD44−); DN4 (CD25−CD44−); ISP (CD25−CD44−CD3ε−CD4−CD8+); DP (CD4+CD8+); ST1 (CD44−NK1.1−); ST2 (CD44hiNK1.1−); ST3 (CD44hiNK1.1+); CD4naive (CD4+CD62LhiCD44−); CD4memory (CD4+CD62L−CD44hi); iNKT (TCRβ+CD1d-αGC+); NKT1a (CD27+IL-2Rβ+); NKT1b (CD27−IL-2Rβ+); NKT2 (CD27+IL-2Rβ−); NKT17 (CD27−IL-2Rβ−); and γδ (γδTCR+).
10
Comprehensive Immune Cell Profiling
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Spleen, MLN, and small intestine lamina propria cells were resuspended in PBS/1% bovine serum albumin and incubated with anti-mouse CD16/CD32 (Mouse BD Fc Block; BD Pharmingen, San Jose, CA, USA) to block non-specific binding sites. For surface staining, cells were incubated with CD4-PerCp-Cy5.5, CD69-APC, CXCR3-PE, CD25-AlexaFluor488, CD69-PE-Cy7, CD11c-PerCp-Cy5.5, CD8α-PE, CD11b-PE-Cy7, CD103-APC, CD11b-PE, CD83-FITC, CD86-APC (eBiosciences, San Diego, CA, USA), T1ST2-FITC (MD Biosciences, St. Paul, MN, USA), or CX3CR1-AlexaFluor488 (R&D Systems, Oxon, UK). Viable cells were distinguished by means of a fixable viability dye eFluor® 780 (eBioscience). For detecting transcription factors, cells were first fixed and permeabilized with Foxp3 Staining Buffer Set (eBioscience) according to manufacturer’s protocol and then stained with Foxp3-APC and RorγT-PE antibodies (eBioscience). Results were collected with BD FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed with FlowLogic software (Inivai Technologies, Mentone, VIC, Australia).
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